The research program of this section of the LESTD seeks to understand the mechanisms and role of bacterial regulation of virulence genes in the pathogenesis of bacterial infection. We use the model system of Bordetella pertussis, a respiratory pathogen, which has an elaborate and refined regulatory system governing its virulence genes. One effort seeks to understand the molecular mechanisms by which the regulator BvgA activates different virulence gene promoters in a time-dependent, or otherwise environmentally-dependent fashion. Another effort seeks to analyze the program expression of virulence genes of this bacterium during the course of infection (in a mouse model). We are also applying our knowledge and experience to develop new and more powerful tools for the genetic analysis and manipulation of bioterrorism threat agent Bacillus anthracis. Regulation of virulence factors in Bordetella pertussis. The expression of virulence factors in the bacterium Bordetella pertussis is regulated in an environmentally responsive fashion under the control of the two-component system encoded by the bvgAS locus. In recent years we have focused research efforts on understanding the mechanisms of gene regulation in this important human pathogen - specifically focussing on the genes encoding pertussis toxin (ptx) and filamentous hemagglutinin (fha). Within the past year we have performed a detailed genetic and biochemical analysis of two regions of the fha promoter, the high affinity primary binding site, and the lower affinity secondary binding region. The primary binding site analysis has helped us to define critical bases involved in DNA recognition and binding by BvgA, and to begin to predict the presence of binding sites in unknown sequences. The secondary binding region analysis revealed that there are no critical bases for BvgA interaction and binding in the context of cooperative interactions with BvgA bound to the primary site. DNA sequence analysis of a large number of random substitutions within either of these regions which were able to restore promoter activity has extended and confirmed these predictions. Recently, powerful biochemical tools have been employed to determine the stoichiometry and configuration of BvgA molecules bound to the promoter DNA of the fha operon. BvgA was substituted at different positions with cysteine and subsequently derivatized with Fe-BABE, a chemical cleavage reagent. Use of these derivatized BvgA molecules has revealed the exact location and orientation of BvgA molecules bound to DNA. These results are consistent with structural studies of NarL, a related response regulator protein from E. coli. Application of the same FeBABE labelling and cleavage techniques to the C-terminal domain of the alpha subunit of RNA polymerase have revealed that BvgA interaction with this key regulatory domain involves a novel form of interaction not previously described for a transcriptional activator. We are currently engaged in survey of the seven most studied and most important virulence gene promoters, using these same affinity cleavage tools to provide a more accurate and detailed view of the BvgA regulon than previously possible. We have also increased our emphasis on the genetic analysis of BvgA, in order to more clearly define the modes of interaction of BvgA with the alpha subunit C-terminal domain. Together, these studies are providing a quantum leap in our understanding of BvgA interaction at virulence gene promoters and have thus increased our understanding of the mechanisms of virulence gene activation in this important human pathogen. In a related project, we have been working on adapting the RIVET (Recombinational In Vivo Expression Technology)system first developed for use in Vibrio cholerae to Bordetella pertussis. This system allows us to determine the level of virulence gene expression at different time points during infection in an animal model. In this way we anticipate gaining insights into the unfolding of the BvgA-mediated regulatory program in real-time during the course of infection. Development of genetic tools for the manipulation of Bacillus anthracis. This project seeks to develop new genetic tools to aid in research into the genetic bases of virulence in Bacillus anthracis. Tools which are required for state of the art genetic analysis would include: transposon mutagenesis, allelic exchange, interspecies (E. coli to B. anthracis) conjugation, genetic (recombinational) mapping, etc. Although some of these tools have been reported in the literature, subsequent attempts to use them have met with variable success. Two major thrusts of the research, based on the current state of the art, are 1)development of allelic exchange vectors which utilize a conditionally countereselectable marker. The ability to select against maintenance of the vector provides the power to force allelic exchange at much greater efficiency than is currently available. 2) To increase the efficiency of transfer of foreign DNA into B. anthracis. In an effort to accomplish this, we have introduced lesions into resident chromosomal genes predicted to encode DNA restriction systems. The resulting mutants are currently being analyzed for their effect on genetic transfer.